ABSTRACT RFA-HL-17-015: Engineered Protein Hydrogels to Modulate Adipose-derived Stromal Cell Secretome and Exosomes for Injectable Myocardial Infarction Therapy Regenerative cell-based therapies have emerged as a promising approach to treat myocardial infarction (MI). However, despite numerous ongoing clinical trials, they have been only mildly successful due to poor cell viability and minimal engraftment. The observed functional improvement has been attributed to paracrine signaling of transplanted cells, which can lead to improved neovascularization. In particular, adipose-derived stromal cells (ASC) are known to secrete a variety of soluble factors that may mediate regeneration. Many injectable biomaterials have been developed to improve regenerative cell-based therapies. Most of these are physical hydrogels that shear-thin and self-heal for ease of injectability. Unfortunately, these hydrogels are often too weak for the demands of an MI application. To address this fundamental need, we develop a new family of injectable biomaterials designed to improve the therapeutic outcome of ASC-based MI therapy. This biomaterial utilizes a novel dynamic covalent chemistry (DCC) crosslinking strategy to create an injectable hydrogel that has the appropriate mechanical integrity for cardiac applications. Additionally, the hydrogel has customizable viscoelastic mechanics, with independently tunable stiffness and stress relaxation properties, to enhance angiogenic paracrine signaling from transplanted cells. Specifically, the material is composed of an engineered elastin-like protein and a chemically modified hyaluronic acid that is networked together through DCC hydrazone bonds to form a biocompatible and enzymatically biodegradable hydrogel. In Specific Aim 1 we evaluate the in vitro ability of the hydrogel to improve ASC viability and enhance their angiogenic paracrine signaling. Rat ASCs will be encapsulated within the engineered hydrogels of varying viscoelastic properties (G' = 0.1, 1, 10 kPa; stress relaxation half-lives = 100, 1000, 10000, ? sec), subjected to an in vitro model of injection, and assayed for membrane damage, metabolic activity, and proliferation. Conditioned media (CM) from ASCs encapsulated within the hydrogels will be collected, and the content of secreted exosomes and the expression of pro-angiogenic factors at both the RNA and protein levels will be quantified. The CM will also be assessed for their functionality via endothelial ?tubule? formation assays with rat endothelial cells. The hydrogel formulation that results in the best angiogenic paracrine signaling will be selected for further in vivo validation in Specific Aim 2, using a rat MI model. Cells will be injected within the best-performing hydrogel into the myocardium, following induction of MI through ligation of the left anterior descending (LAD) artery (106 cells in 75 L of material per animal). Comparison groups include sham, saline only, saline with cells, and hydrogel only. Bioluminescence and fluorescence imaging (days 0, 1, 3, 7,14, 21, 28) will determine the integrity and viability of transplanted cells and material, respectively, and functional recovery after MI will be assessed using echocardiography (days 7, 28) and hemodynamic measurements (day 28). Heart explants will be analyzed for evidence of necrosis, inflammation, tissue regeneration, and presence of transplanted cells (day 28).